Translation initiation in eukaryotes involves recruitment by mRNAs of the 40S ribosomal subunit and other components of the translation machinery at either the 5′ cap-structure or an internal ribosome entry site (IRES). Following its recruitment, the 40S subunit moves to an initiation codon. One widely held notion of translation initiation postulates that the 40S subunit moves from the site of recruitment to the initiation codon by scanning through the 5′ leader in a 5′ to 3′ direction until the first AUG codon that resides in a good nucleotide context is encountered (Kozak “The Scanning Model for Translation: An Update” J. Cell Biol. 108:229-241 (1989)). More recently, it has been postulated that translation initiation does not involve scanning, but may involve tethering of ribosomal subunits at either the cap-structure or an IRES, or clustering of ribosomal subunits at internal sites (Chappell et al. “Ribosomal shunting mediated by a translational enhancer element that base pairs to 18S rRNA” PNAS USA 103(25):9488-9493 (2006); Chappell et al., “Ribosomal tethering and clustering as mechanisms for translation initiation” PNAS USA 103(48):18077-82 (2006)). The 40S subunit moves to an accessible AUG codon that is not necessarily the first AUG codon in the mRNA. Once the subunit reaches the initiation codon by whatever mechanism, the initiator Methionine-tRNA, which is associated with the subunit, base-pairs to the initiation codon, the large (60S) ribosomal subunit attaches, and peptide synthesis begins.
Inasmuch as translation is generally thought to initiate by a scanning mechanism, the effects on translation of AUG codons contained within 5′ leaders, termed upstream AUG codons, have been considered, and it is known that an AUG codon in the 5′ leader can have either a positive or a negative effect on protein synthesis depending on the gene, the nucleotide context, and cellular conditions. For example, an upstream AUG codon can inhibit translation initiation by diverting ribosomes from the authentic initiation codon. However, the notion that translation initiates by a scanning mechanism does not consider the effects of potential initiation codons in coding sequences on protein synthesis. In contrast, the tethering/clustering mechanisms of translation initiation suggests that putative initiation codons in coding sequences, which include both AUG codons and non-canonical codons, may be utilized, consequentially lowering the rate of protein synthesis by competing with the authentic initiation codon for ribosomes.
Micro RNA (miRNA)-mediated down-regulation can also negatively impact translation efficiency. miRNAs are generally between 21-23 nucleotides in length and are components of ribonucleoprotein complexes. It has been suggested that miRNAs can negatively impact protein levels by base-pairing to mRNAs and reducing mRNA stability, nascent peptide stability and translation efficiency (Eulalio et al. “Getting to the Root of miRNA-Mediated Gene Silencing” Cell 132:9-14 (1998)). Although miRNAs generally mediate their effects by base-pairing to binding sites in the 3′ untranslated sequences (UTRs) of mRNAs, they have been shown to have similar repressive effects from binding sites contained within coding sequences and 5′ leader sequences. Base-pairing occurs via the so-called “seed sequence,” which includes nucleotides 2-8 of the miRNA. There may be more than 1,000 different miRNAs in humans.
The negative impact of putative initiation codons in mRNA coding sequences and miRNA-binding sites in mRNAs pose challenges to the pharmaceutical industry. For example, the industrial production of protein drugs, DNA vaccines for antigen production, general research purposes and for gene therapy applications are all affected by a sub-optimal rate of protein synthesis or sequence stability. Improving protein yields and higher protein concentration can minimize the costs associated with industrial scale cultures, reduce costs of producing drugs and can facilitate protein purification. Poor protein expression limits the large-scale use of certain technologies, for example, problems in expressing enough antigen from a DNA vaccine to generate an immune response to conduct a phase 3 clinical trial.